The present invention relates to a linear water-soluble quaternary ammonium polymer, a process for the preparation of this polymer, the use of this polymer as a displacer in the displacement mode of chromatography and a method for separating biomolecules through the displacement mode chromatography using this polymer.
The majority of the separation or isolation procedures used in the pharmaceutical field, or other related fields such as the biochemical, the biotechnological and the chemical fields, rely on chromatography, i.e. a differential separation between two phases, a stationary one (usually solid) and a mobile one (usually fluid).
Elution mode chromatography is the most commonly used mode of chromatography. In this method, the sample containing different components to be separated is adsorbed on the stationary phase. The mobile phase, called eluent, is chosen such that the components bind reversibly onto the stationary phase. As the eluent is flowing over the stationary phase, the different components migrate along the column at a speed which reflects their relative affinity for the stationary phase.
Biomolecules such as proteins are generally purified by gradient elution mode chromatography using ion-exchange adsorbents as stationary phase. The elution involves changing the pH of the buffer solution passing through the column or, more common, increasing the salt concentration in the buffer solution passing through the column. When the pH of the solution is changed, the electric charges on the proteins or the ion-exchange adsorbent material are changed and the proteins are released. With an increase of the salt concentration, the salts weaken the bonds between the proteins and the ion-exchange adsorbent material to release the proteins. As the salt level is gradually increased, the proteins having the smaller number of charges, respectively the lower charge density (mass to charge ratio) will generally be released and eluted first and those with the larger number of charges, respectively the higher charge density, will be released later.
Displacement chromatography is a special mode of chromatography. The basic principle of chromatography still applies, but in this case the driving force behind the separation is the push of an advancing front of a so-called displacer, rather than the (increasing) elution strength of a mobile phase. Under the influence of an advancing displacer front, the substance mixture is resolved into consecutive zones of the pure substances.
The displacer is a substance with a higher binding affinity to the stationary phase than the components to be separated. As the displacer front advances, the number of stationary sites available to the binding components are continuously decreased, which engenders competition for the binding sites between the displacer and the components and also between the components themselves. Under ideal conditions, the more strongly bond ones displace the more weakly bond ones until all substances are focused into consecutive individual zones of pure substance that leave the column at the speed of the advancing displacer front in the order of the stationary phase affinities.
One of the important distinctions between displacement and elution mode chromatography is that, in displacement mode chromatography, the displacer front always remains behind the consecutive feed zones, while, in elution mode chromatography, the eluent or desorbent moves through the feed zones with the components to be separated.
The displacer is a unique feature of displacement mode chromatography. At the same time, the choice of the displacer has consequences not only for the success, but also for the economic soundness of the final method. Mathematical simulations, which take into account concentrations in the mobile and stationary phases, allow some predictions concerning the necessary displacer characters for a given substance mixture to be separated.
A displacement mode chromatography method for purifying proteins using, as a stationary phase, a cation-exchange resin and, as a displacer, a cationic species is disclosed in U.S. Pat. No. 5,478,924. These cationic species are poly(quaternary ammonium) salts having a dendritic framework formed by reiterative reaction sequences starting from pentaerythritol. Their structure consists, on the one hand, in a hydrophobic interior zone containing ramifications connected to the initial core pentaerythritol and, on the other hand, in a hydrophilic exterior surface region bearing the quaternary ammonium groups.
The first and second generation pentaerythritol-based dendrimers, all terminated with trimethylammonium groups, having respectively a molar mass of about 2,000 g/mol and 6,000 g/mol, are described as being effective for the purification of a two-protein mixture, i.e. xcex1-chymotrypsinogen A and cytochrome C.
However, it is mentioned that, due to the presence of impurities in the first generation dendrimers contributing to the desorption of the proteins and depression of their isotherms, the cytochrome C zone is considerably less concentrated in relation to the xcex1-chymotrypsinogen A zone.
The preparation of these poly(quaternary ammonium) salts is relatively tedious, time and cost consuming, involving low overall yield multi-step synthesis. The level of purity and homogeneity cannot be high, particularly for the high molar mass second generation pentaerythritol-based dendrimers.
It is also reported that the xe2x80x9czeroxe2x80x9d generation pentaerythritol-based dendrimers, terminated with trimethylammonium groups and having for instance a molecular weight of 620 g when X=Cl, are equally effective for the separation of the two proteins.
However, S. D. Gadam and S. M. Cramer have reported, in Chromatographia, 1994, 39, 409-18, that the behaviour of such low molar mass displacers depends to a much higher degree on the chromatographic conditions, for example the salt content of the mobile phase. As a consequence, the switch from displacer to elution promoter is more likely in the case of these substances.
One of the aims of the present invention is to provide a low molar mass polymer capable to be used as a cationic displacer, i.e. having high solubility in an aqueous carrier with a high binding tendency towards a cation-exchange stationary phase. This particular polymer has to be easily available on a large scale with high level of purity and homogeneity.
To this effect, the present invention relates to a linear water-soluble quaternary ammonium polymer having a homopolymeric chain of repeating unit of general formula (I): 
where each of R1 and R2 independently represents a member selected from the group consisting of linear or branched alkyl, hydroxyalkyl, alkoxyalkyl groups having from 1 to 6 carbon atoms, Xxe2x88x92 represents an anion, n is an integer comprised between 30 and 220. Furthermore, the polymer has a molar mass distribution of less than or equal to 1.5.
The anion represented by Xxe2x88x92 is selected from the group consisting of Fxe2x88x92, Clxe2x88x92, Brxe2x88x92, NO3xe2x88x92, OHxe2x88x92, HSO4xe2x88x92, xc2xd SO42xe2x88x92, CH3COOxe2x88x92.
The preferred linear water-soluble quaternary ammonium polymer has the above general formula (I), where both R1 and R2 represent a methyl group and Xxe2x88x92 represents Clxe2x88x92, n being an integer comprised between 30 and 220. Such a preferred polymer has a number average molar mass comprised between about 4,800 g/mol and about 35,000 g/mol and a molar mass distribution of less than or equal to 1.5.
Since the 1950s, a number of preparations of linear water-soluble quaternary ammonium polymers having a homopolymeric chain of the same above-mentioned repeating unit have been described. They are based on polymerisation reactions employing various initiation methods, including radically, ionically, or X-ray induced polymerisation, and involving an alternating intra-intermolecular chain propagation.
However, most of these syntheses are oriented towards high molar mass polymers having a number average molar mass of up to 200,000 g/mol, or even more, with a conversion rate of more than 95%, which means that almost all the starting monomer is consumed in the course of the reaction. From these syntheses, result generally polymers having molar mass distributions greater than 3.
Such high molar mass polymers are produced, for example, using a technology as it is described in FR 2 448 546 and the classical polymerisation conditions to be applied to obtain such polymers are described for instance in J. Ulbricht, xe2x80x9cGrundlagen der Synthese von Polymerenxe2x80x9d, 2nd edition, Huethig and Wepf, 1992. It is reported in this textbook that to produce low molar masses, one has to increase the free-radical polymerisation initiator concentration and/or the reaction temperature.
In the particular case of the polymerisation of diallyldimethylammonium chloride, H. Dautzenberg et al. report, in xe2x80x9cPolyelectrolytes: Fornation, characterisation and applicationxe2x80x9d, Carl Hanser Verlag, Munich, 1994, p. 20, that increasing the initiator concentration and the temperature as proposed above leads to an increase of linear propagation with the consequence of pendent double bounds and in the following chain branching.
To obtain a linear polymer having a narrow molar mass distribution, the radically initiated polymerisation reaction is normally only to low conversion, i.e. less than 10%.
None of the existing preparations offers a method to obtain, without special fractionation, a poly(diallyldimethylammonium chloride)-type polymer having a low molar mass and a narrow molar mass distribution at conversions more than 40%.
Another of the aims of the present invention is to provide a method for the preparation of the polymer as defined above by the general formula (I) and having a molar mass distribution of less than or equal to 1.5.
To this effect, the present invention relates to a process for the preparation of the above mentioned polymer of general formula (I).
In this process, a quaternary ammonium monomer of general formula (II): 
where each of R1 and R2 independently represents a member selected from the group consisting of linear or branched alkyl, hydroxyalkyl, alkoxyalkyl groups having from 1 to 6 carbon atoms and Xxe2x88x92 represents an anion;
is brought into contact with a catalytic amount of a free-radical polymerisation initiator in an oxygen-free aqueous reaction medium at a temperature comprised in the range of 30xc2x0 C. and 70xc2x0 C., said monomer being introduced into the reaction medium in such a way that its concentration in said reaction medium in the course of the polymerisation reaction is less than or equal to 3 mol/l.
Preferably, the anion represented by Xxe2x88x92 is selected from the group consisting of Fxe2x88x92, Clxe2x88x92, Brxe2x88x92, NO3xe2x88x92, OHxe2x88x92, HSO4xe2x88x92, xc2xd SO42xe2x88x92, CH3COOxe2x88x92.
Preferably, the monomer is continuously introduced into the reaction medium in such a rate that its concentration in the reaction medium remains above the lower limit of 1 mol/l. More preferably, the monomer is continuously introduced into the reaction medium in order to maintain constant its concentration in the reaction medium.
Preferably, the reaction medium is maintained at a constant temperature.
The free-radical polymerisation initiator used in the process is a water soluble peroxy initiator. Preferably, this initiator is selected from the group consisting of ammonium peroxydisulfate, potassium peroxydisulfate, sodium peroxydisulfate, lithium peroxydisulfate. More preferably, the initiator is ammonium peroxydisulfate.
The free-radical polymerisation initiator is used as such. It does not require the use of any auxiliary agent such as metal sequestering agents.
The concentration of the free-radical polymerisation initiator in the reaction medium is comprised between 1.10xe2x88x923 mol/l and 50.10xe2x88x923 mol/l.
Furthermore, the reaction medium can be filtrated under ultrafiltration conditions in order to separate the obtained polymer from non-reacted monomer and initiator. The use of a membrane allowing a cut-off of 3,000 g/mol is appropriate.
In another aspect, the present invention relates to the use of the above mentioned polymer of general formula (I), as a cation-exchange displacer for a displacement mode chromatography method involving a cation-exchange stationary phase.
In a further aspect, the present invention relates to a method for separating biomolecules contained in a sample by using the above mentioned polymer of general formula (I). This method comprises:
i) passing said sample containing the biomolecules to be separated through cation-exchange stationary phase so that said biomolecules adsorb on said stationary phase;
ii) passing an aqueous solution of said polymer through said stationary phase so that the biomolecules to be separated are displaced by said polymer by displacement mode chromatography.
This method is appropriate for the separation of molecules bearing a permanent or temporary positive net-charge or positively charged patches, which enable them to interact with the cation-exchange stationary phase.
Such molecules, with the required above-mentioned electrolytic character, can be biomolecules, synthetic or semi-synthetic molecules such as amino-acids, peptides, proteins, nucleosides, nucleotides, polynucleotides, alkaloids, antibiotics, saccharides, polysaccharides, lipids, drugs, drugs candidates and derivatives thereof.
The appropriate stationary phase used in the method can be any type of weak or strong cation-exchange stationary phase. Such stationary phases can be those bearing on their surface carboxymethyl groups, sulphopropyl groups, methyl-sulphonate groups, phospho groups or any carboxylate and sulfo groups anchored by any other spacers, or other groups bearing permanent or pH-dependent negative charges. These stationary phases can also be stationary phases displaying a partial cation exchanger character, such as hydroxy- and fluoroapatite and mixed mode phases.
The aqueous solution of the polymer is prepared before passing through the stationary phase. This solution can eventually contain water-miscible organic solvents.
As the polymer is a quaternary ammonium salt, it can be considered as being not dependent on the pH. Therefore the aqueous solution and the mobile phase can be any buffer usually suitable for cation-exchange chromatography, including volatile buffer systems. Under certain circumstances, buffer usually suitable for anionic ion-exchange chromatography can also be used.
The preferred buffer substances are citric acid, acetic acid, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), N,N-bis(2-hydroxyethyl)glycine (BICINE), phosphate, xcex1,xcex1,xcex1-tris(hydroxymethyl)-methylamine (TRIS).